Project Summary/Abstract The podocyte is the primary glomerular cell targeted for injury during the development and progression of glomerular diseases that eventuate in end stage renal disease (ESRD). Renal expression of cyclooxygenase-2 (COX-2) increases in models of progressive renal injury, and selective COX-2 inhibitors decrease proteinuria and retard progressive renal injury in models of renal ablation, diabetes and salt- sensitive hypertension. However, the mechanisms by which inhibition of prostaglandin synthesis can ameliorate progressive renal injury remain undetermined. We propose that local expression of COX-2 in podocytes predisposes to progressive glomerular injury. We and others have demonstrated a selective increase of COX-2 expression in podocytes in models of progressive glomerular injury and found that over- expression of COX-2 in podocytes renders the glomerulus susceptible to adriamycin nephropathy. However, the mechanisms underlying COX-2 up-regulation in podocytes and potential roles in podocyte function in response to injury remain incompletely understood. We hypothesize increased podocyte COX-2 generates metabolites that interact with the local podocyte RAS, predisposing the podocyte to further injury in response to renal insults. The goal of these studies is to examine the potential role of prostaglandins as mediators and/or modulators of podocyte injury during the development of diabetic nephropathy. We propose two complementary specific aims. In Specific Aim I, we will investigate the role of altered COX-2 expression in podocyte injury during diabetic nephropathy in vivo, using genetically engineered mice that either selectively overexpress COX-2 or have genetic deletion of COX-2 in podocytes. We will also utilize mice with genetic deletions of specific prostanoid receptors to determine which prostaglandin(s) mediate podocyte injury and will elucidate interactions between podocyte COX-2 and the local podocyte renin-angiotensin system (RAS) during the development of diabetic nephropathy. In Specific Aim II, we will investigate the mechanisms by which COX-2 increases podocyte injury in response to high glucose in vitro using conditionally immortalized mouse podocytes from wild type mice, from the transgenic mice overexpressing COX-2 or null for COX-2 expression in podocytes. We will investigate the effects of high glucose on 1) the signals leading to increased podocyte COX-2 expression; 2) the effects of alterations in COX-2 expression on structure and function of differentiated podocytes in response to high glucose; and 3) the role of podocyte COX-2 as a mediator of increased production of ROS in podocytes. Although COX-2 inhibitors effectively ameliorate renal injury in animal models, the awareness of their potential to promote thrombotic vascular injury and to raise blood pressure preclude their clinical use in patients with progressive renal disease. Identification of which prostaglandins mediate glomerular injury and the determination of their mechanisms of injury may allow the development of therapeutic strategies with fewer potential side effects.